Zika virus (ZIKV) has recently caused explosive outbreaks in the Americas and is unexpectedly associated with congenital microcephaly and other fetal abnormalities as well as Guillain Barr{tilde over (e)} syndrome (Schuler-Faccini et al., 2016). ZIKV was first isolated from a sentinel rhesus macaque in 1947 in the Zika Forest of Uganda (Dick et al., 1952). Human ZIKV infections have only sporadically been detected for decades. However, since 2007, ZIKV has rapidly spread across islands in the South Pacific and into the Americas, causing the outbreak on Yap Island in Micronesia, a subsequent outbreak in French Polynesia, and explosive, widespread epidemics in the Americas (Petersen et al., 2016). The World Health organization (http://www.who.int/emergencies/zika-virus/situation-report/6-october-2016/en/) has reported over 73 countries and territories with active ZIKV outbreaks/epidemics. Despite urgent medical needs, neither clinically approved vaccine nor antiviral is available for prevention and treatment.
ZIKV is a mosquito-borne member from the genus flavivirus within the family Flaviviridae. Besides ZIKV, many other flaviviruses are significant human pathogens, including the four serotypes of dengue (DENV-1 to -4), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses. Flaviviruses have a positive-sense single-stranded RNA genome approximately 11,000 nucleotides in length. The genome contains a 5′ untranslated region (UTR), single open-reading frame (ORF), and 3′ UTR. The ORF encodes three structural (capsid [C], precursor membrane [prM], and envelope [E]) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The structural proteins form virus particles and function in virus entry into cells. The nonstructural proteins participate in viral replication, virion assembly, and evasion of host innate immune responses (Lindenbach, 2013). Like other flaviviruses, ZIKV enters cells through the receptor-mediated endocytosis. After low pH-induced fusion with the endosome membrane, flaviviruses release and translate their genomic RNA in the endoplasmic reticulum (ER). Viral RNA replication occurs in the virus-induced replication complexes formed in the ER membrane. Progeny viruses form on the ER-derived membrane as immature virus particles, in which prM/E heterodimers form trimeric spikes with icosahedral symmetry. After removal of the pr from the prM by host furin protease during the transit through the Golgi network, the immature, non-infectious virions become mature infectious viruses. Finally, progeny virions are released through an exocytosis pathway (Lindenbach, 2013). Rapid progress has been made on ZIKV research in the past two years, including the high-resolution structures of virus (Kostyuchenko et al., 2016; Sirohi et al., 2016), reverse genetic systems (Atieh et al., 2016; Schwarz et al., 2016; Shan et al., 2016b; Tsetsarkin et al., 2016; Weger-Lucarelli et al., 2017; Xie et al., 2016), animal models (Lazear et al., 2016; Rossi et al., 2016), and vaccine development (Abbink et al., 2016; Dowd et al., 2016; Larocca et al., 2016).
Development of an effective and affordable ZIKV vaccine is a public health priority. Multiple strategies have been taken, including DNA- or viral vector-expressing subunit, chimeric, and live-attenuated vaccines (Dawes et al., 2016). Three frontrunner candidates, including two DNA vaccines expressing viral structural proteins prM and E (Dowd et al., 2016; Larocca et al., 2016) and one purified inactivated ZIKV vaccine (PIV) based on Puerto Rico strain PRVABC59 (Abbink et al., 2016), protect monkeys from ZIKV challenge. These frontrunners are currently in phase I clinical trials.
The present invention addresses the need for technologies that can increase the yield of virus production to improve accessibility of inactivated vaccines and reduce costs without compromising vaccine immunogenicity.